| Shock is a pathologic process of metabolic and cellular injury caused by insufficience of tissue perfusion,while fluid resuscitation is one of the important corresponding treatments.Increasing evidence suggests that HMGB1 is an important mediators involved in inflammatory responses.HMGB1 is not only a late mediator released by macrophages in sepsis,but also a kind of alarmin,as an early mediator,which takes part in the occurrence of traumatic,hemorrhagic shock and other non-infectious inflammation.Meanwhile,more and more reports show that deacetylase SIRT1 is involved in the regulation of inflammatory responses,relieving multiple organ damage induced by ischemia-reperfusion and sepsis.However,SIRT1 is rarely reported for the deacetylation of HMGB1,and the study of the SIRT1-deacetylated HMGB1 signaling pathway in the specific disease model of shock is relatively insufficient.We hope to identify the role of HMGB 1 acetylation in the development of severe shock,by starting with the de-acetylation of SIRT1.Methods There will be three sections in this study:clinical,animal and cell.A.In the clinical study,serum HMGB1 content was measured in 18 patients with hemorrhagic shock at different time points,which was compared with the same period of healthy volunteers;At the same time,18 patients with hemorrhagic shock was graded with APACHEII,and sperman method was used to analyze the rank relation of HMGB1 peak.B.In the animal study,96 rats were used to reproduce HS/R model for detecting the levels of HMGB1 in rat serum and various organs at different time points,and the most representative time point(4h)and organ(kidney)were selected for the following-up study;the distribution of HMGB1 in the kidney and the level of acetylation were measured at 4h after HS/R;the expression and activity of SIRT1 in renal tubular epithelial cells of HS/R rats and the interaction with HMGB1 were detected;HMGB1 neutralizing antibody and PD was applied as treatment to rats after HS/R and the level of HMGB1,inflammatory factors and SIRT1 were measured;the effect of SIRT1 on HMGB1 distribution and acetylation level was detected;the over all level of rat renal function,apoptosis and overall survival time during the activation of SIRT1 by PD in rats after HS/R.C.In the cytology experiment,the HK-2 model was used to explore the effect of different H/R time on cell viability;the distribution of HMGB1 and the level of acetylation in H/R cells were detected;the expression and activity of SIRT1 in H/R cells and the interaction with HMGB 1 were detected;transient transfection method was applied to overexpress SIRT1 and SIRT1H363Y(a kind of deacetylase domain mutation,which resulted in a significant decrease in SIRT1 deacetylase activity)in HK-2 cells and compared with the Control group to determine whether the regulation level of SIRT1 on HMGB1 depends on the deacetylation activity of SIRT1;the effect of interfering sirt1 on the distribution of HMGB 1 and the content of supernatant in HK-2 cells after H/R stimulation were detected by small interfering RNA technique;the acetylated antibodies of K28,K29,K30,K90 and K177 were designed to detect the specific modification sites of HMGB1 deacetylated by SIRT1;the effect of PD on the activation of SIRT1 and the effect of the distribution and the acetylation level of HMGB1 will be investigated in HK-2 cells of H/R.Results Section A.In the clinical study,HMGB1 level in the serum of patients with acute hemorrhagic shock increased at a peak of 371.6± 69.4(ng/mL)at 4 h,then gradually decrease;The peak of HMGB1 content in serum was positively correlated with the order of APACHEII score,and the spearman correlation coefficient r2=0.76.Section B.In the experimental part with the rats,the contents of HMGB1 in the kidney,liver,lung and intestine tissues were decreased.In order to focus on the relationship between the release of HMGB1 and the content of serum,the kidney was selected as the object of study.The total amount of HMGB1 in the kidney tissue decreased by 94.6 ± 11.8(ng/mL)compared with the Sham group at 2 h after HS/R;The serum HMGB1 increased by 290.4±13.3(ng/mL)at 4h compared with Sham group.In the early stage of HS/R,the total content of HMGB1 in the kidney was decreased.In contrast,the content of HMGB1 in the cytoplasm was increased,and the level of acetylation was also increased to a large extent;The expression of SIRT1 was decreased in the rat renal tubular epithelial cells after hemorrhagic shock,and the activity of SIRT1 was more significant.At the same time,the interaction between SIRT1 and HMGB1 was enhanced;HMGB1 neutralizing antibody reduced the level of HMGB 1 and the level of inflammatory factors in serum of HS/R rats,but it failed to prevent the HMGB 1 nucleus shift phenomenon in its solid organ cells and prevent its release,but with the use of polydatin,the results can be effectively reversed;PD could increase the expression of SIRT1 in the kidney of HS/R rats and significantly improve the activity of SIRT1;after the activation of SIRT1 by PD,the HMGB1 acetylation degree is greatly reduced with more remained in solid organ cells;at the same time,the renal tubular apoptosis and renal function were improved,and the survival time was prolonged.Section C.The results of cytology experiments showed that under the conditions with 6h absence of oxygen and 2h of reoxygenation,there is increasing HMGB1 acetylation level in HK-2 cells,while HMGB1 tends to shift from the nucleus to the cytoplasm,and then to culture supernatant;the expression of SIRT1 in HK-2 cells was decreased,and the activity of SIRT1 was significantly decreased,while the interaction between SIRT1 and HMGB1 was enhanced during H/R;SIRT1H363Y could not reduce HMGB1 acetylation levels in HK-2 cells and their release into culture medium,but the overexpression of SIRT1 could reduce HMGB1 acetylation levels and their release into culture medium;the SiRNA of SIRT1 may significantly increase the level of HMGB1 acetylation in HK-2 cells after H/R;The K90 and K177 sites are particularly evident changed;PD could increase SIRT1 expression in HK-2 cells stimulated by H/R and also improve SIRT1 activity significantly;SIRT1 activation inhibited HMGB1 in HK-2 cells translocated from nuclear-cytoplasm-supernatant.Conclusions1.HMGB1 peaks were found in the serum of patients 4h after hemorrhagic shock,while the higher the peak value,the higher the death risk.2.The increase of serum HMGB 1 in the early stage of hemorrhagic shock was related to its release from solid organ cells such as kidney,and the active release of HMGB1 was mainly mediated by acetylation from nucleus to cytoplasm.3.SIRT1 activity(deacetylation)decreased in shock,causing the enhancentment of HMGB1 acetylation,which led HMGB1 translocation from the nucleus to the cytoplasm and the final release.4.SIRT1 and HMGB1 have natural interactions(immunoprecipitation),the main role is in HMGB1 lysine residues K90 and K177 sites.5.Early release of hemorrhagic shock HMGB1 results in systemic inflammatory response and local organ damage,while SIRT1 activator can increase the activity of renal SIRT1,reduce the acetylation of HMGB1 and renal apoptosis,and eventually restore renal function.SIRT1 activator can also prolong the survival time of hemorrhagic shock animals.Therefore,the decrease of SIRT1 activity plays an important role in the acetylation of HMGB1 followed by release from the organ cell in hemorrhagic shock.Activation of SIRT1 may be served as a novel approach for the treatment of severe shock. |
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